This Proposal is designed to address: 1) an important disease that needs novel drugs - no new drugs for Mycobacterium tuberculosis in 40 years and multi-drug resistant strains as well as extreme drug resistant strains are becoming more common;2) the lack of structural information for an entire class of drug targets, the membrane proteins - less than 1% of known protein structures are membrane proteins, while 25 to 30% of the genome of most organisms code for membrane proteins - in addition, membrane proteins are more frequently effective drug targets than water-soluble proteins;3) biological, functional and structural characterization of validated targets - we will characterize only those proteins that are essential for Mtb growth and by targeting membrane proteins, especially the outer membrane proteins, access to the drug targets will not require transport across the bacterial membranes. 4) a gap in screening technology - new small molecule screening technologies based on solution and solid state NMR spectroscopy will be developed specifically for membrane proteins. We have developed an Initial Target List from preliminary results and from literature on essential genes, virulence factors, identification of outer membrane proteins and numerous individual studies on specific potential targets. Some of these proteins are already validated as high potential pharmaceutical targets, these form a Prioritized Target List that will allow all of the Projects and Cores to initiate their efforts on the first day of funding. From biological function (Project 1) to assay development (Project 2) to structural characterization (Project 3) these activities will work closely together. Assays coupled with molecular structure will help establish structure-activity-relationships. Assay development will enable screening against small molecules important for understanding function and potentially important for structural studies. The assays we develop and the ligands we identify will fuel biological experiments designed to understand the life and infection cycle of Mtb. These ligands will be useful as lead compounds in drug discovery, and while this is beyond the scope of this Program, this team will protect the intellectual property for those who may want to pursue the development of drugs for these membrane protein targets. To accomplish these goals a unique team of investigators has been brought together with extensive knowledge of: Mycobacterium tuberculosis, essential Mtb genes, membrane protein physiology, molecular biology, biophysics, and structural characterization. The Program offers access to two of the premier NMR facilities in the world along with their expertise in methodology and technology development, in addition, the Team brings with it unique expression libraries of Mtb membrane proteins and the superb screening facilities and expertise of the Burnham Institute. PROJECT 1: TARGET IDENTIFICATION (NIEDERWEIS, M) PROJECT 1 DESCRIPTION (provided by applicant): More than 60% of all drug targets are membrane proteins. However, only one of the current antituberculous drugs, ethambutol, targets membrane proteins of M. tuberculosis, indicating that this vast potential is largely untapped for M. tuberculosis. The recent discovery of a new diarylquinoline drug that targets the ATP synthase of M. tuberculosis and exceeds the activities of the two main TB drugs isoniazid and rifampin provides proof of principle that this assumption is correct. Therefore, we have selected 11 out of approximately 800 putative inner membrane proteins of M. tuberculosis, which are essential for growth of M. tuberculosis in vitro or in mice. These proteins were selected to cover a broad variety of functions, such as transport of essential nutrients, cell division, export of proteins and cell wall and lipid biosynthesis. Furthermore, they are sufficiently small to be amenable to current solid-state NMR methods. Outer membrane proteins offer the tremendous advantage as drug targets that inhibitors may not need to cross the outer membrane which is an extremely efficient permeability barrier in mycobacteria. In addition, they are likely to represent novel drug targets because they do not appear to show any similarity to other proteins. Therefore, we included seven putative outer membrane proteins in our list of initial targets, six of which have no known function. The goal of this project is to validate the potential of the 18 selected membrane proteins as drug targets, to elucidate the physiological function of the six proteins with no known homologs and to identify putative interaction partners for some of these proteins.